Detection of Brain Injury

ABSTRACT

The present invention provides minimally invasive methods of detecting, diagnosing, and assessing neuronal damage associated with traumatic brain injury (TBI) or chronic traumatic encephalopathy (CTE). Specific species of microRNAs (miRNA), small, noncoding RNA molecules that play gene regulatory functions, are correlated with cellular damage and oxidative stress following TBI or CTE, allowing for rapid, minimally-invasive diagnosis and assessment of brain injury. The early identification and longitudinal assessment of neuronal damage in subjects suffering from or at risk of suffering from a TBI (e.g., football players, boxers, military personnel, fall victims) will improve clinical outcomes by guiding critical medical and behavioral decision making.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Patent Application Ser. No. 61/970,613, filed Mar. 26, 2014,the disclosure of which is incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

This application includes as part of the originally filed subject mattera Sequence Listing electronically submitted via EFS-Web as a single textfile named “UM014002SL.txt”. The Sequence Listing text file was createdon Mar. 25, 2015 and is 122 kb in size. The contents of the SequenceListing are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The acute and chronic molecular effects of mild TBI (mTBI) have not beenwell studied or characterized. Over the past several decades it hasbecome increasingly clear that repetitive mTBI is capable of alteringthe biochemical activity of the brain in ways that cannot be detected bycurrent methodologies. Highlighting this issue is the definitive linkbetween repeated mTBI and the development of chronic traumaticencephalopathy (CTE) in athletes and soldiers. The immediate issuefacing an individual that has suffered a mTBI is determining when it issafe to return to high risk activities after a concussive injury withoutrisking permanent brain damage that occurs at a cellular level.Unfortunately, no non-invasive diagnostic methods or tools currentlyexist to evaluate TBI-caused neuronal damage or CTE progression.

MicroRNAs (“miRNAs”) are endogenous, non-coding small RNAs approximately22 base pair in length. MiRNAs are highly conserved across species,accounting for 1-2% of the genes in eukaryotic genomes while potentiallyregulating 30% of all annotated human genes. Mature miRNAs bindsequence-specific sites in the 3′-untranslated region (3′-UTR) of theirtarget mRNAs and inhibit protein synthesis by repressing translation orregulating mRNA degradation. Some single miRNA have been predicted toregulate several hundred-target mRNAs. MiRNAs are important epigeneticregulators of biological processes and many are expressed specificallyin an organ, cell or cellular compartment. The discovery thatcirculating miRNAs are altered in pathological conditions has spawnedthe development of miRNAs as potential biomarkers of neurodegenerativediseases. The release of miRNAs whether passive, associated withArgonaut2 (ago2) or mediated by active secretion via exosomes ormicrovesicles is believed to dramatically effect protein expressionthroughout the central nervous system. In the case of CTE, thedefinitive diagnosis of the disease is made post-mortem by theidentification of neuronal death in specific areas of the brain e.g.,cerebral hemispheres, thalamus and medial temporal lobe. Howeverprofound loss of neurons and brain atrophy are late-occurring events inthe pathogenesis of the disease and are preceded by metabolic changessuch as hyperphosphorylation of tau and deposition of neurofibrillarytangles presumably leading to synaptic dysfunction and loss, neuriteretraction and axonal degeneration. Such damage has been demonstrated torelease stable miRNA into the systemic circulation.

SUMMARY OF THE INVENTION

The present invention features methods and kits useful for the minimallyinvasive detection of brain injury. In a first aspect, the inventionprovides a method of detecting a brain injury in a patient, such as ahuman, by contacting a biological sample derived from the patient withat least one miR-specific oligodeoxynucleotide probe having at least 70%complementarity to a sequence selected from SEQ ID NOs. 1-69,determining the expression level of at least one microRNA represented bySEQ ID NOs. 1-69 by quantifying at least one such miR-specificoligodeoxynucleotide probe, and comparing the expression level with acontrol expression level derived from a healthy subject, wherein a 1.2fold or greater difference between the patient and control microRNAexpression levels indicates that the patient has suffered a braininjury. In one embodiment, the method further provides for the treatmentof the patient with a therapeutically-effective amount of anantioxidant, such as alpha-tocopherol, ascorbate, coenzyme Q,alpha-lipoic acid, curcumin, glutathione, uric acid, a carotene,superoxide dismutase, a catalase, a peroxiredoxin, a thioredoxin,tirilazad mesylate, or NXY-059, if brain injury is detected. In anotherembodiment, the biological sample is blood, cerebral spinal fluid, braintissue. In a further embodiment, the biological sample is blood plasmaor serum. The method can be used to detect brain injuries such astraumatic brain injury and chronic traumatic encephalopathy. The methodcan be performed using polymerase chain reaction (PCR), in situhybridization, Northern blot, or gene chip analysis using, e.g., DNAoligonucleotide probes. In one embodiment, the biological sample isderived before the patient has suffered a brain injury. In anotherembodiment, the method is repeated on biological samples derived fromthe patient over a period of time to allow for measurement of braininjury progression or healing.

In a second aspect, the invention provides a minimally-invasive methodof detecting a brain injury in a patient, such as a human, by contactinga blood, plasma, or serum sample derived from the patient with at leastone miR-specific oligodeoxynucleotide probe having at least 70%complementarity to a sequence selected from SEQ ID NOs. 1-69,determining the expression level of at least one microRNA represented bySEQ ID NOs. 1-69 by quantifying at least one such miR-specificoligodeoxynucleotide probe, and comparing the expression level with acontrol expression level derived from a healthy subject, wherein a 1.2fold or greater difference between the patient and control microRNAexpression levels indicates that the patient has suffered a braininjury. In one embodiment, the method further provides for the treatmentof the patient with a therapeutically-effective amount of anantioxidant.

In a third aspect, the invention provides a kit detecting a brain injurythat includes (a) one or more miR-specific oligonucleotide probes havingat least 70% complementarity to a sequence selected from SEQ ID NOs.1-69, (b) one or more control samples, and (c) instructions indicatingthe use of the probes and control samples for detecting a brain injury.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

As used herein, the singular form “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a cell” includes a plurality of cells, including mixturesthereof. The term “a nucleic acid molecule” includes a plurality ofnucleic acid molecules.

As used herein, the terms below have the meanings indicated.

The term “bond” refers to a covalent linkage between two atoms, or twomoieties when the atoms joined by the bond are considered to be part oflarger substructure. A bond may be single, double, or triple unlessotherwise specified. A dashed line between two atoms in a drawing of amolecule indicates that an additional bond may be present or absent atthat position.

An “expression profile” or “hybridization profile” of a particularsample is essentially a fingerprint of the state of the sample; whiletwo states may have any particular gene similarly expressed, theevaluation of a number of genes simultaneously allows the generation ofa gene expression profile that is unique to the state of the cell. Thatis, normal tissue may be distinguished from abnormal (e.g., diseased orinjured) tissue, and within abnormal tissue, different prognosis states(for example, good or poor long term survival prospects) may bedetermined. By comparing expression profiles of tissue (e.g., blood,tissue biopsy or necropsy sample, or cerebral spinal fluid) in differentstates, information regarding which genes are important (including bothup- and down-regulation of genes) in each of these states is obtained.The identification of sequences that are differentially expressed intissue, as well as differential expression resulting in differentprognostic outcomes, allows the use of this information in a number ofways. For example, a particular treatment regime may be evaluated (e.g.,to determine whether a therapeutic drug acts to improve the long-termprognosis in a particular patient). Similarly, diagnosis may be done orconfirmed by comparing patient samples with known expression profiles.Furthermore, these gene expression profiles (or individual genes) allowscreening of drug candidates that alter or normalize tissue expressionprofiles to impart a clinical benefit.

The term “imaging agent” as used herein refers to any moiety useful forthe detection, tracing, or visualization of a compound when coupledthereto. Imaging agents include, e.g., an enzyme, a fluorescent label(e.g., fluorescein), a luminescent label, a bioluminescent label, amagnetic label, a metallic particle (e.g., a gold particle), ananoparticle, an antibody or fragment thereof (e.g., a Fab, Fab′, orF(ab′)₂ molecule), and biotin. An imaging agent can be coupled to acompound by, for example, a covalent bond, ionic bond, van der Waalsinteraction or a hydrophobic bond. An imaging agent can be a radiolabelcoupled to or a radioisotope incorporated into the chemical structure ofa compound used according to the invention. Methods of detecting suchimaging agents include, but are not limited to, positron emissiontomography (PET), X-ray computed tomography (CT) and magnetic resonanceimaging (MRI).

As used herein interchangeably, a “miR gene product,” “microRNA,” “miR,”or “miRNA” refers to the unprocessed (e.g., precursor) or processed(e.g., mature) RNA transcript from a miR gene. As the miR gene productsare not translated into protein, the term “miR gene products” does notinclude proteins. The unprocessed miR gene transcript is also called a“miR precursor” or “miR prec” and typically comprises an RNA transcriptof about 70-100 nucleotides in length. The miR precursor can beprocessed by digestion with an RNAse (for example, Dicer, Argonaut, orRNAse III (e.g., E. coli RNAse III)) into an active 19-25 nucleotide RNAmolecule. This active 19-25 nucleotide RNA molecule is also called the“processed” miR gene transcript or “mature” miRNA.

The term “neurodegenerative disorder” as used herein, refers to anydisease, disorder, condition, or symptom characterized by the structuralor functional loss of neurons. Neurodegenerative disorders include,e.g., Alzheimer's disease, Parkinson's disease, Huntington's Disease,Lewy Body dementia, and amyotrophic lateral sclerosis (ALS).

As used herein, “probe oligonucleotide” or “probe oligodeoxynucleotide”refers to an oligonucleotide that is capable of hybridizing to a targetoligonucleotide. By “miR-specific oligonucleotide probe” or “probeoligonucleotide specific for a miR” is meant a probe oligonucleotidethat has a sequence selected to hybridize to a specific miR geneproduct, or to a reverse transcript of the specific miR gene product.

“Target oligonucleotide” or “target oligodeoxynucleotide” refers to amolecule to be detected (e.g., via hybridization).

As used herein, “sample” refers to any biological matter derived from asubject (e.g., a human). Samples include, but are not limited to, blood,PBMC, plasma, platelets, serum, cerebral spinal fluid (CSF), saliva,cells, tissues, and organs. In certain embodiments of the invention,preferred samples include blood plasma, CSF, and brain tissue.

The phrase “therapeutically effective” is intended to qualify the amountof active ingredients used in the treatment of a disease or disorder.This amount will achieve the goal of reducing or eliminating the diseaseor disorder.

The term “therapeutically acceptable” refers to those compounds (orsalts, esters, prodrugs, tautomers, zwitterionic forms, etc. thereof)which are suitable for use in contact with the tissues of patientswithout undue toxicity, irritation, and allergic response, arecommensurate with a reasonable benefit/risk ratio, and are effective fortheir intended use.

As used herein, reference to “treatment” of a patient is intended toinclude prophylaxis. The term “patient” means mammals and non-mammals.Mammals means any member of the mammalian class including, but notlimited to, humans; non-human primates such as chimpanzees and otherapes and monkey species; farm animals such as cattle, horses, sheep,goats, and swine; domestic animals such as rabbits, dogs, and cats;laboratory animals including rodents, such as rats, mice, and guineapigs; and the like. Examples of non-mammals include, but are not limitedto, birds, and the like. The term “patient” does not denote a particularage or sex.

The term “prodrug” refers to a compound that is made more active invivo. Certain compounds may also exist as prodrugs, as described inHydrolysis in Drug and Prodrug Metabolism: Chemistry, Biochemistry, andEnzymology, Testa, Bernard and Wiley-VHCA, Zurich, Switzerland 2003.Prodrugs of the compounds are structurally modified forms of thecompound that readily undergo chemical changes under physiologicalconditions to provide the compound. Additionally, prodrugs can beconverted to the compound by chemical or biochemical methods in an exvivo environment. For example, prodrugs can be slowly converted to acompound when placed in a transdermal patch reservoir with a suitableenzyme or chemical reagent. Prodrugs are often useful because, in somesituations, they may be easier to administer than the compound, orparent drug. They may, for instance, be bio-available by oraladministration whereas the parent drug is not. The prodrug may also haveimproved solubility in pharmaceutical compositions over the parent drug.A wide variety of prodrug derivatives are known in the art, such asthose that rely on hydrolytic cleavage or oxidative activation of theprodrug. An example, without limitation, of a prodrug is a compound thatis administered as an ester (the “prodrug”), but then is metabolicallyhydrolyzed to the carboxylic acid, the active entity. Additionalexamples include peptidyl derivatives of a compound.

Compounds can exist as therapeutically acceptable salts. Suitable saltsinclude those formed with both organic and inorganic acids. Such acidaddition salts will normally be pharmaceutically acceptable. However,salts of non-pharmaceutically acceptable salts may be of utility in thepreparation and purification of the compound in question. Basic additionsalts may also be formed and be pharmaceutically acceptable. For a morecomplete discussion of the preparation and selection of salts, refer toStahl, P. Heinrich, Pharmaceutical Salts: Properties, Selection, andUse, Wiley-VCHA, Zurich, Switzerland (2002).

The term “therapeutically acceptable salt” as used herein, representssalts or zwitterionic forms of a compound which are water or oil-solubleor dispersible and therapeutically acceptable as defined herein. Thesalts can be prepared during the final isolation and purification of thecompounds or separately by reacting the appropriate compound in the formof the free base with a suitable acid. Representative acid additionsalts include acetate, adipate, alginate, L-ascorbate, aspartate,benzoate, benzenesulfonate (besylate), bisulfate, butyrate, camphorate,camphorsulfonate, citrate, digluconate, formate, fumarate, gentisate,glutarate, glycerophosphate, glycolate, hemisulfate, heptanoate,hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethansulfonate (isethionate), lactate, maleate, malonate,DL-mandelate, mesitylenesulfonate, methanesulfonate,naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate,pamoate, pectinate, persulfate, 3-phenylproprionate, phosphonate,picrate, pivalate, propionate, pyroglutamate, succinate, sulfonate,tartrate, L-tartrate, trichloroacetate, trifluoroacetate, phosphate,glutamate, bicarbonate, para-toluenesulfonate (p-tosylate), andundecanoate. Also, basic groups in the compounds can be quaternized withmethyl, ethyl, propyl, and butyl chlorides, bromides, and iodides;dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl,myristyl, and steryl chlorides, bromides, and iodides; and benzyl andphenethyl bromides. Examples of acids which can be employed to formtherapeutically acceptable addition salts include inorganic acids suchas hydrochloric, hydrobromic, sulfuric, and phosphoric, and organicacids such as oxalic, maleic, succinic, and citric. Salts can also beformed by coordination of the compounds with an alkali metal or alkalineearth ion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows System xCT staining in sham and TBI-injured brains. Panel Ais a surgical sham animal. Panel B is a TBI animal 48 hours after TBI.Panel C is a TBI animal 46 days post-TBI. Panel D is a graphicalrepresentation of the loss of and modest recovery of xCT straining thatoccurs over a 46 day period after TBI. One way ANOVA, Tukey's post-hoc.**=p<0.01. Data collected by laser scanning cytometry.

FIG. 2 panels E and F show neurological severity scores and foot faults,respectively, from injured and un-injured animals at 48 hours, 2 weeks,and 46 days post-TBI. In both assessments there was a significantimprovement from 48 hours to 46 days post-TBI that corresponds with thereturn of xCT to the neuromotor cortex. However, neurological scoringremained significantly lower than shams that had normal levels of xCTexpression. n=12 animals per group; unpaired two-tailed t-test.***=p<0.001.

FIG. 3 shows System xCT (red) and GFAP (green) staining in the cortex ofrats and human patients. Panel A is a surgical sham rat. Panel B is aTBI rat 46 days after injury. Panel C is a human control patient. PanelD is a stage IV CTE patient. Data collected at 60× using an OlympusFV1000 confocal microscope. Human tissue was kindly provided by theCenter for the Study of Traumatic Encephalopathy (Boston University).

FIG. 4 is a heat map displaying fold changes in 84 oxidative stressgenes comparing TBI to Sham. Total RNA was isolated from FFPE 7 μMslices from 4 sham control rats or 4 TBI rats and pooled for cDNAsynthesis and preamp with universal oxidative stress array RT²PCRprimers. Oxidative Stress Array plates were run on a Bio-Rad iQ5iCycler. Data for control and TBI was normalized with Rplpl. Boundarywas set for 2-fold changes.

FIG. 5 is a scatter plot displaying fold changes in predicted xCTtargeting miRNA. Total RNA was isolated from FFPE 7 μM slices from 4sham control rats or 4 TBI rats and pooled for cDNA synthesis and preampwith universal oxidative stress array RT²PCR primers. Rat miFinder Arrayplates were run on a Bio-Rad iQ5 iCycler. Data for control and TBI wasnormalized with SNORD61 and SNORD95. Boundary was set for 2-foldchanges.

FIG. 6 is a chart of miRNA probes used in PCR Array CMIHS02277.*=predicated to target xCT (SLC7A11).

FIG. 7 is a chart showing the fold change, 95% confidence interval, andp values between miRNA expression profiles on the PCR Array CMIHS02277between human peripheral blood plasma obtained from control subjects andthose having suffered acute TBI (within 24-72 hours).

FIG. 8 is a chart showing the fold change, 95% confidence interval, andp values between miRNA expression profiles on the PCR Array CMIHS02277between human peripheral blood plasma obtained from control subjects andfootball players.

FIG. 9 is a chart showing the fold change, 95% confidence interval, andp values between miRNA expression profiles on the PCR Array CMIHS02277between human peripheral blood plasma obtained from football players andthose having suffered acute TBI (within 24-72 hours).

FIG. 10 is a chart showing the fold change, 95% confidence interval, andp values between miRNA expression profiles on the PCR Array CMIHS02277between human peripheral blood plasma obtained from control subjects andsoccer players.

FIG. 11 is a chart showing the fold change, 95% confidence interval, andp values between miRNA expression profiles on the PCR Array CMIHS02277between human peripheral blood plasma obtained from control subjects andthose with chronic TBI.

FIG. 12 is a chart showing the fold change, 95% confidence interval, andp values between miRNA expression profiles on the PCR Array CMIHS02277between human peripheral blood plasma obtained from subjects that havesuffered acute TBI (within 24-72 hours) and those with chronic TBI.

DETAILED DESCRIPTION OF THE INVENTION

The invention features non-invasive methods of detecting, diagnosing,and tracking traumatic brain injury (TBI) or chronic traumaticencephalopathy (CTE), and related conditions, by evaluating theexpression of one or more microRNAs (“miRNAs”) in a sample (e.g., braintissue, blood sample, or cerebral spinal fluid sample) derived from asubject (e.g., a human) considered to have suffered from, or is at riskof suffering from, a TBI or other neurological defect. The methods ofthe invention can be used to diagnose, predict, and monitor theprogression of neuronal damage caused by single or multiple traumaticevents, such as those experienced in, e.g., falling accidents, activesports (e.g., boxing and football), vehicular or labor accidents, or bylaw enforcement or military personnel. In one embodiment of theinvention, the methods of the invention allow for the detection of miRNAcorrelated with TBI neuronal damage in the blood plasma of a subjectthat has or as at risk of experiencing a traumatic event.

The present invention embraces the discovery that certain miRNA speciesare differentially regulated following neuronal damage. Tables 1 and 2list miRNA species (H. sapiens and R. norvegicus, respectively) found tobe upregulated or downregulated in R. norvegicus that experience TBI ina laboratory neuronal damage model.

TABLE 1 miRNA Name H. sapiens 5′ Sequence SEQ ID NO. H. sapiens 3′Sequence SEQ ID NO. miR-142 cauaaaguagaaagcacuacu 1uguaguguuuccuacuuuaugga 37 miR-21 uagcuuaucagacugauguuga 2caacaccagucgaugggcugu 38 let-7a ugagguaguagguuguauaguu 3cuauacaaucuacugucuuuc 39 let-7b ugagguaguagguugugugguu 4cuauacaaccuacugccuuccc 40 let-7f ugagguaguagauuguauaguu 5 miR-144ggauaucaucauauacuguaag 6 uacaguauagaugauguacu 41 miR-150ucucccaacccuuguaccagug 7 cugguacaggccugggggacag 42 miR-32uauugcacauuacuaaguugca 8 caauuuagugugugugauauuu 43 miR-130auucacauugugcuacugucugc 9 cagugcaauguuaaaagggcau 44 miR-101caguuaucacagugcugaugcu 10 uacaguacugugauaacugaa 45 miR-18auaaggugcaucuagugcagauag 11 acugcccuaagugcuccuucugg 46 let-7dagagguaguagguugcauaguu 12 cuauacgaccugcugccuuucu 47 miR-181baacauucauugcugucggugggu 13 cucacugaacaaugaaugcaa 48 miR-223cguguauuugacaagcugaguu 14 ugucaguuugucaaauacccca 49 miR-320aaaagcuggguugagagggcga 15 miR-374 uuauaauacaaccugauaagug 16cuuaucagauuguauuguaauu 50 let-7e ugagguaggagguuguauaguu 17cuauacggccuccuagcuuucc 51 miR-196b uagguaguuuccuguuguuggg 18ucgacagcacgacacugccuuc 52 miR-96 uuuggcacuagcacauuuuugcu 19aaucaugugcagugccaauaug 53 miR-423 ugaggggcagagagcgagacuuu 20agcucggucugaggccccucagu 54 miR-210 agccccugcccaccgcacacug 21cugugcgugugacagcggcuga 55 miR-182 uuuggcaaugguagaacucacacu 22ugguucuagacuugccaacua 56 miR-196a uagguaguuucauguuguuggg 23cggcaacaagaaacugccugag 57 miR-39-3p ucaccggguguaaaucagcuug 58 miR-9ucuuugguuaucuagcuguauga 24 auaaagcuagauaaccgaaagu 59 miR-133aagcugguaaaauggaaccaaau 25 uuugguccccuucaaccagcug 60 miR-30auguaaacauccucgacuggaag 26 cuuucagucggauguuugcagc 61 miR-137uuauugcuuaagaauacgcguag 27 miR-23a gggguuccuggggaugggauuu 28aucacauugccagggauuucc 62 miR-25 aggcggagacuugggcaauug 29cauugcacuugucucggucuga 63 miR-32 uauugcacauuacuaaguugca 30caauuuagugugugugauauuu 64 miR-203a gugaaauguuuaggaccacuag 31 miR-153ucauuuuugugauguugcagcu 32 uugcauagucacaaaagugauc 65 miR-218a-luugugcuugaucuaaccaugu 33 augguuccgucaagcaccaugg 66 miR-26auucaaguaauccaggauaggcu 34 ccuauucuugguuacuugcacg 67 miR-148aaaaguucugagacacuccgacu 35 ucagugcacuacagaacuuugu 68 miR-19aaguuuugcauaguugcacuaca 36 ugugcaaaucuaugcaaaacuga 69

TABLE 2 miRNA Name R. norvegicus 5′ Sequence SEQ ID NO. R. norvegicus 3′Sequence SEQ ID NO. miR-142 cauaaaguagaaagcacuacu 70uguaguguuuccuacuuuaugga 106 miR-21 uagcuuaucagacugauguuga 71caacagcagucgaugggcuguc 107 let-7a ugagguaguagguuguauaguu 72cuauacaaucuacugucuuucc 108 let-7b ugagguaguagguugugugguu 73cuauacaaccuacugccuuccc 109 let-7f ugagguaguagauuguauaguu 74cuauacaaucuauugccuucc 110 miR-144 ggauaucaucauauacuguaagu 75uacaguauagaugauguacu 111 miR-150 ucucccaacccuuguaccagug 76cugguacaggccuggggga 112 miR-32-5p uauugcacauuacuaaguugca 77gcaauuuagugugugugauauu 113 miR-130a gcucuuuucacauugugcuacu 78cagugcaauguuaaaagggcau 114 miR-101a ucaguuaucacagugcugaugc 79uacaguacugugauaacugaa 115 miR-18a uaaggugcaucuagugcagauag 80acugcccuaagugcuccuucu 116 let-7d agagguaguagguugcauaguu 81cuauacgaccugcugccuuucu 117 miR-181b aacauucauugcugucggugggu 82cucacugaacaaugaaugcaa 118 miR-223 cguguauuugacaagcugaguug 83ugucaguuugucaaauacccc 119 miR-320 gccuucucuucccgguucuucc 84aaaagcuggguugagagggcga 120 miR-374 auauaauacaaccugcuaagug S5cuuagcacguuguauuauuauu 121 let-7e ugagguaggagguuguauaguu 86cuauacggccuccuagcuuucc 122 miR-196b uagguaguuuccuguuguuggg 87ucgacagcacgacacugccuuca 123 miR-96 uuuggcacuagcacauuuuugcu 88caaucaugugcagugccaauau 124 miR-423 ugaggggcagagagcgagacuuuu 89agcucggucugaggccccucagu 125 miR-210 agccacugcccacagcacacug 90cugugcgugugacagcggcuga 126 miR-182 uuuggcaaugguagaacucacaccg 91 miR-196auagguaguuucauguuguuggg 92 ucggcaacaagaaacugccuga 127 miR-39 miR-9aucuuugguuaucuagcuguauga 93 auaaagcuagauaaccgaaagu 128 miR-133aagcugguaaaauggaaccaaau 94 uuugguccccuucaaccagcug 129 miR-30auguaaacauccucgacuggaag 95 cuuucagucggauguuugcagc 130 miR-137acggguauucuuggguggauaa 96 uuauugcuuaagaauacgcguag 131 miR-23agggguuccuggggaugggauuu 97 aucacauugccagggauuucc 132 miR-25aggcggagacacgggcaauugc 98 cauugcacuugucucggucuga 133 miR-32uauugcacauuacuaaguugca 99 gcaauuuagugugugugauauu 134 miR-203aagugguucuuaacaguucaac 100 gugaaauguuuaggaccacuag 135 miR-153gucauuuuugugauguugcagcu 101 uugcauagucacaaaagugauc 136 miR-218a-1uugugcuugaucuaaccaugu 102 aaacaugguuccgucaagcac 137 miR-26auucaaguaauccaggauaggcu 103 ccuauucuugguuacuugcac 138 miR-148gaaguucuguuauacacucagg 104 ucagugcaucacagaacuuugu 139 miR-19aucguuuugcauaguugcacu 105 ugugcaaaucuaugcaaaacuga 140Methods of miRNA Expression Profiling

The expression level of at least one miRNA species can be measured in abiological sample (e.g., an organ, tissue, or cell sample, such as braintissue, blood sample, or cerebral spinal fluid (CSF)) obtained from apatient (e.g., a human). For example, a tissue sample (e.g., braintissue, blood, or CSF) can be removed from a patient suspected ofsuffering from or at risk of suffering a brain injury (e.g., TBI or CTE)by conventional biopsy techniques. In another embodiment, a blood or CSFsample can be removed from the patient (e.g., a human), and cells (e.g.,white blood cells) or serum can be isolated for RNA extraction bystandard techniques. In order to determine baseline miRNA expressionprofiles, a blood, CSF, or tissue sample is preferably obtained from thepatient prior to initiation of any activity that carries a heightenedrisk of TBI, including but not limited to impact sports (e.g., boxing,American football, rugby, hockey, baseball, and soccer), military or lawenforcement service, medical conditions that leave subjects susceptibleto falls (e.g., blindness, advanced age), or any other that places thesubject at increased risk of suffering TBI (e.g., race car driving,skydiving, and victims of assault). Baseline blood or tissue samples arealso ideally obtained prior to radiotherapy, chemotherapy or othertherapeutic treatment in order to gauge miRNA expression profile changesduring the course of treatment. A corresponding control tissue or bloodsample can be obtained from unaffected tissues of the patient, from anormal human individual or population of normal individuals, or fromcultured cells corresponding to the majority of cells in the patient'ssample. The control tissue or blood sample is then processed along withthe sample from the patient, so that the miRNA expression profilederived from the patient's sample can be compared to a correspondingmiRNA expression profile derived from a sample taken from a controlsubject or group. A reference miRNA expression profile standard for thebiological sample can also be used as a control.

An alteration (e.g., an increase or decrease) in the level of one ormore of the miRNAs identified herein (e.g., SEQ ID NOS:1-140) in thesample obtained from a patient (e.g., a human), relative to the level ofcorresponding miRNAs in a control sample, is indicative of the presenceof brain injury (e.g., TBI) in the patient. In one embodiment, theexpression level of at least one miRNA in the test sample is greaterthan the expression level of a corresponding miRNA in the control sample(i.e., expression of the miRNA is “up-regulated”). As used herein,expression of a miRNA is “up-regulated” when the amount of miRNA in afluid, cell, or tissue sample from a patient is greater than the amountof the same miRNA in a control fluid, cell, or tissue sample. In anotherembodiment, the expression level of the at least one miRNA in the testsample is less than the expression level of the corresponding miRNA inthe control sample (i.e., expression of the miRNA is “down-regulated”).As used herein, expression of a miRNA is “down-regulated” when theamount of miRNA produced in a fluid, cell, or tissue sample from apatient is less than the amount produced in a fluid, control cell, ortissue sample. A patient miRNA expression profile is considered toindicate the presence of a brain injury if the up or down-regulation is1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 fold or greater relativeto the control expression profile. The relative miRNA expression in thecontrol and normal samples can be determined with respect to one or moremiRNA expression standards. The standards can comprise, for example, azero miRNA gene expression level, the miRNA expression profiles ofstandardized cell lines, the miRNA expression profiles in unaffectedtissues of the patient (e.g., a human), or the average level of miRNAexpression previously obtained for a population of normal controls(e.g., human controls).

The level of a miRNA expression in a sample can be measured using anytechnique that is suitable for detecting RNA expression levels in abiological sample. Suitable techniques (e.g., Northern blot analysis,RT-PCR, in situ hybridization) for determining RNA expression levels ina biological sample (e.g., cells, tissues) are well known to those ofskill in the art. In a particular embodiment, the level of at least onemiRNA species is detected using Northern blot analysis. For example,total cellular RNA can be purified from cells by homogenization in thepresence of nucleic acid extraction buffer, followed by centrifugation.Nucleic acids are precipitated, and DNA is removed by treatment withDNase and precipitation. The RNA molecules are then separated by gelelectrophoresis on agarose gels according to standard techniques, andtransferred to nitrocellulose filters. The RNA is then immobilized onthe filters by heating. Detection and quantification of specific RNA isaccomplished using appropriately labeled DNA or RNA probes complementaryto the RNA in question. See, e.g., Molecular Cloning: A LaboratoryManual, J. Sambrook et al., eds., 2nd edition, Cold Spring HarborLaboratory Press, 1989, Chapter 7, the entire disclosure of which isincorporated by reference.

Suitable probes for Northern blot hybridization of a given miRNA can beproduced from the nucleic acid sequences of the miRNA sequencesdescribed and listed herein and include, but are not limited to, probeshaving at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or completecomplementarity to a miRNA of interest. Methods for preparation oflabeled DNA and RNA probes, and the conditions for hybridization thereofto target nucleotide sequences, are described in Molecular Cloning: ALaboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold SpringHarbor Laboratory Press, 1989, Chapters 10 and 11, the disclosures ofwhich are incorporated herein by reference. For example, the nucleicacid probe can be labeled with, e.g., a radionuclide, such as ³H, ³²P,³³P, ¹⁴C or ³⁵S; a heavy metal; a ligand capable of functioning as aspecific binding pair member for a labeled ligand (e.g., biotin, avidinor an antibody); a fluorescent molecule; a chemiluminescent molecule; anenzyme or the like.

Probes can be labeled to high specific activity by either the nicktranslation method of Rigby et al. (1977), J. Mol. Biol. 113:237-251 orby the random priming method of Fienberg et al. (1983), Anal. Biochem.132:6-13, the entire disclosures of which are incorporated herein byreference. The latter is the method of choice for synthesizing³²P-labeled probes of high specific activity from single-stranded DNA orfrom RNA templates. For example, by replacing preexisting nucleotideswith highly radioactive nucleotides according to the nick translationmethod, it is possible to prepare ³²P-labeled nucleic acid probes with aspecific activity well in excess of 10⁸ cpm/microgram. Autoradiographicdetection of hybridization can then be performed by exposing hybridizedfilters to photographic film. Densitometric scanning of the photographicfilms exposed by the hybridized filters provides an accurate measurementof miR gene transcript levels. Using another approach, miR genetranscript levels can be quantified by computerized imaging systems,such as the Molecular Dynamics 400-B 2D Phosphorimager available fromAmersham Biosciences, Piscataway, N.J.

Where radionuclide labeling of DNA or RNA probes is not practical, therandom-primer method can be used to incorporate an analogue, forexample, the dTTP analogue5-(N—(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl)deoxyuridinetriphosphate, into the probe molecule. The biotinylated probeoligonucleotide can be detected by reaction with biotin-bindingproteins, such as avidin, streptavidin, and antibodies (e.g.,anti-biotin antibodies) coupled to fluorescent dyes or enzymes thatproduce color reactions.

In addition to Northern and other RNA hybridization techniques,determining the levels of RNA transcripts can be accomplished using thetechnique of in situ hybridization. This technique requires fewer cellsthan the Northern blotting technique, and involves depositing wholecells onto a microscope cover slip and probing the nucleic acid contentof the cell with a solution containing radioactive or otherwise labelednucleic acid (e.g., cDNA or RNA) probes. This technique is particularlywell suited for analyzing tissue biopsy samples from subjects. Thepractice of the in situ hybridization technique is described in moredetail in U.S. Pat. No. 5,427,916, the entire disclosure of which isincorporated herein by reference. Suitable probes for in situhybridization of a given miRNA can be produced from the nucleic acidsequences having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%or complete complementarity to a miRNA of interest, as described above.

The relative number of miRNA gene transcripts in cells can also bedetermined by reverse transcription of miRNA gene transcripts, followedby amplification of the reverse-transcribed transcripts by polymerasechain reaction (RT-PCR). The levels of miRNA gene transcripts can bequantified in comparison with an internal standard, for example, thelevel of mRNA from a “housekeeping” gene present in the same sample. Asuitable “housekeeping” gene for use as an internal standard includes,e.g., myosin or glyceraldehyde-3-phosphate dehydrogenase (G3PDH).Methods for performing quantitative and semi-quantitative RT-PCR, andvariations thereof, are well known to those of skill in the art.

In some instances, it may be desirable to simultaneously determine theexpression level of a plurality of different miRNA species in a sample(e.g., brain tissue, blood, or cerebral spinal fluid (CSF)). In otherinstances, it may be desirable to determine the expression level of thetranscripts of all known miRNA species correlated with a brain injury.Assessing brain injury-specific expression levels for hundreds of miRNAspecies is time consuming and requires a large amount of total RNA(e.g., at least 20 micrograms for each Northern blot) andautoradiographic techniques that require radioactive isotopes.

To overcome these limitations, an oligolibrary, in microchip format(i.e., a microarray), may be constructed containing a set ofoligonucleotide (e.g., oligodeoxynucleotides) probes that are specificfor a set of miRNA species. Using such a microarray, the expressionlevel of multiple microRNAs in a biological sample (e.g., brain tissue,blood, or cerebral spinal fluid (CSF)) can be determined by reversetranscribing the RNAs to generate a set of target oligodeoxynucleotides,and hybridizing them to probe the oligonucleotides on the microarray togenerate a hybridization, or expression, profile. The hybridizationprofile of the test sample can then be compared to that of a controlsample to determine which microRNAs have an altered expression levelconsistent with a suspected disease, condition, or disorder, such astraumatic brain injury.

Accordingly, the invention provides methods of diagnosing whether asubject has, or is at increased risk of suffering from a TBI comprisingreverse transcribing RNA from a test sample (e.g., brain tissue, blood,or cerebral spinal fluid (CSF)) obtained from the subject (e.g., ahuman) to provide a set of target oligodeoxynucleotides, hybridizing thetarget oligodeoxynucleotides to a microarray comprising miRNA-specificprobe oligonucleotides to provide a hybridization profile for the testsample, and comparing the test sample hybridization profile to ahybridization profile generated from a control sample or referencestandard, wherein an alteration in the signal of at least one miRNA isindicative of the subject either having, or being at risk fordeveloping, TBI. In one embodiment, the microarray comprisesmiRNA-specific probe oligonucleotides for a substantial portion of allknown human miRNAs. In one embodiment, the microarray comprisesmiRNA-specific probe oligonucleotides for one or more miRNAs selectedfrom the group consisting of miR-142, miR-21, let-7a, let-7b, let-7f,miR-144, miR-150, miR-32, miR-130a, miR-101a, miR-18a, let-7d, miR-181b,miR-223, miR-320, miR-374, let-7e, miR-196b, miR-96, miR-423, miR-210,miR-182, miR-196a, miR-39, miR-9a, miR-133a, miR-30a, miR-137, miR-23a,miR-25, miR-32, miR-203a, miR-153, miR-218-1, miR-26a, miR-148a, andmiR-19a.

The microarray can be prepared from gene-specific oligonucleotide probesgenerated from known miRNA sequences. The array may contain twodifferent oligonucleotide probes for each miRNA, one containing theactive, mature sequence and the other being specific for the precursorof the miRNA. The array may also contain controls, such as one or moremouse sequences differing from human orthologs by only a few bases,which can serve as controls for hybridization stringency conditions.tRNAs or other RNAs (e.g., rRNAs, mRNAs) from both species may also beprinted on the microchip, providing an internal, relatively stable,positive control for specific hybridization. One or more appropriatecontrols for non-specific hybridization may also be included on themicrochip. For this purpose, sequences are selected based upon theabsence of any homology with any known miRNAs.

The microarray may be fabricated using techniques known in the art. Forexample, probe oligonucleotides of an appropriate length, e.g., 40nucleotides, are 5′-amine modified at position C6 and printed usingcommercially available microarray systems, e.g., the GeneMachineOmniGrid™ 100 Microarrayer and Amersham CodeLink™ activated slides.Labeled cDNA oligomer corresponding to the target RNAs is prepared byreverse transcribing the target RNA with labeled primer. Following firststrand synthesis, the RNA/DNA hybrids are denatured to degrade the RNAtemplates. The labeled target cDNAs thus prepared are then hybridized tothe microarray chip under hybridizing conditions, e.g., 6×SSPE/30%formamide at 25° C. for 18 hours, followed by washing in 0.75× TNT (TrisHCl/NaCl/Tween 20) at 37° C. for 40 minutes. At positions on the arraywhere the immobilized probe DNA recognizes a complementary target cDNAin the sample, hybridization occurs. The labeled target cDNA marks theexact position on the array where binding occurs, allowing automaticdetection and quantification. The output consists of a list ofhybridization events, indicating the relative abundance of specific cDNAsequences, and therefore the relative abundance of the correspondingcomplementary miRNAs, in the patient sample. Image intensities of eachspot on the array are proportional to the abundance of the correspondingmiRNA in the patient sample.

The use of the array has several advantages for miRNA expressiondetection. First, the global expression of several hundred genes can beidentified in the same sample at one time point. Second, through carefuldesign of the oligonucleotide probes, expression of both mature andprecursor molecules can be identified. Third, in comparison withNorthern blot analysis, the chip requires a small amount of RNA, andprovides reproducible results using 2.5 micrograms of total RNA. Therelatively limited number of miRNAs (a few hundred per species) allowsthe construction of a common microarray for several species, withdistinct oligonucleotide probes for each. Such a tool would allow foranalysis of trans-species expression for each known miRNA under variousconditions.

In addition to use for quantitative expression level assays of specificmiRNA, a microchip containing miRNA-specific probe oligonucleotidescorresponding to a substantial portion of the miRNome, preferably theentire miRNome, may be employed to carry out miRNA gene expressionprofiling, for analysis of miRNA expression patterns. Distinct miRNAsignatures can be associated with established disease markers, ordirectly with a disease state.

According to the expression profiling methods described herein, totalRNA from a sample (e.g., brain tissue, blood, or cerebral spinal fluid(CSF)) from a subject (e.g., a human) suspected of suffering or at riskof suffering a TBI is quantitatively reverse transcribed to provide aset of labeled target oligodeoxynucleotides complementary to the RNA inthe sample. The target oligodeoxynucleotides are then hybridized to amicroarray comprising miRNA-specific probe oligonucleotides to provide ahybridization profile for the sample. The result is a hybridizationprofile for the sample representing the expression pattern of miRNA inthe sample. The hybridization profile comprises the signal from thebinding of the target oligodeoxynucleotides from the sample to themiRNA-specific probe oligonucleotides in the microarray. The profile maybe recorded as the presence or absence of binding (signal vs. zerosignal). More preferably, the profile recorded includes the intensity ofthe signal from each hybridization. The profile is compared to thehybridization profile derived from a normal, i.e., non-TBI, controlsample. An alteration in the signal is indicative of the presence of, orpropensity to develop, TBI in the subject.

Other techniques for measuring miRNA gene expression are also within theskill in the art, and include various techniques for measuring rates ofRNA transcription and degradation.

Treatment of Brain Injury

As described herein, brain injury is associated with marked loss ofSystem x_(c) ⁻ antiporter expression in brain tissues and an overallloss in antioxidant capacity in these tissues. Weak antioxidantmechanisms allow for accumulation of reactive oxygen species (ROS) thatchemically damage surround cells and tissues.

Upon making a clinical determination that a patient (e.g., a human) hassuffered a brain injury, a clinician may determine that administrationof an antioxidant or antioxidant therapy course is appropriate. Examplesof antioxidants include, but are not limited to, alpha-tocopherol,ascorbate, coenzyme Q, alpha-lipoic acid, curcumin, glutathione, uricacid, carotenes (e.g., retinol, beta-carotene), superoxide dismutase,catalases, peroxiredoxins, thioredoxins, tirilazad mesylate, andNXY-059. In one embodiment, the patient is administered atherapeutically-effective amount of one or more antioxidants in order toslow the progression of brain injury.

Methods of Diagnostic Imaging

The present invention provides for the diagnosis and medical evaluationof patients (e.g., a human) suffering from, or at risk of suffering fromTBI, CTE, or related conditions. For example, an imaging agent specificfor System x_(c) ⁻ can also be used, alone or in combination with otheragents and compounds, in medical imaging applications to diagnose orfollow the progression of diseases, disorders, conditions or symptomsrelated to TBI or CTE in a patient (e.g., a human). For example,radiologists and other medical clinicians are skilled in the use ofradiographic imaging devices, such as positron emission tomography (PET)scanners, and methods of imaging tracer compounds, such as theradionuclides. (e.g., Saha, Basics of PET Imaging: Physics, Chemistry,and Regulations, Springer (2010) ISBN 978-1-4419-0804-9, herebyincorporated by reference).

The methods of the present invention are also useful for the medicalimaging and diagnosis of humans and animals, e.g., domesticated animal,companion animals (e.g., dogs and cats), exotic animals, farm animals(e.g., ungulates, including horses, cows, sheep, goats, and pigs), andanimals used in scientific research (e.g., rodents and non-humanprimates).

Compound Administration and Formulation

Basic addition salts can be prepared during the final isolation andpurification of the compounds by reaction of a carboxy group with asuitable base such as the hydroxide, carbonate, or bicarbonate of ametal cation or with ammonia or an organic primary, secondary, ortertiary amine. The cations of therapeutically acceptable salts includelithium, sodium, potassium, calcium, magnesium, and aluminum, as well asnontoxic quaternary amine cations such as ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, diethylamine, ethylamine, tributylamine, pyridine,N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine,dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine,1-ephenamine, and N,N-dibenzylethylenediamine. Other representativeorganic amines useful for the formation of base addition salts includeethylenediamine, ethanolamine, diethanolamine, piperidine, andpiperazine.

A salt of a compound can be made by reacting the appropriate compound inthe form of the free base with the appropriate acid. A compound can beprepared in a form of pharmaceutically acceptable salts that will beprepared from nontoxic inorganic or organic bases including but notlimited to aluminum, ammonium, calcium, copper, ferric, ferrous,lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc,and the like. Salts derived from pharmaceutically acceptable organicnon-toxic bases include salts of primary, secondary, and tertiaryamines, substituted amines including naturally-occurring substitutedamines, cyclic amines, and basic ion exchange resins, such as arginine,betaine, caffeine, choline, ethylamine, 2-diethylaminoethano,1,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,histidine, hydroxylamine, isopropylamine, lysine, methylglucamine,morpholine, piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, trishydroxylmethyl aminomethane, tripropyl amine, and tromethamine.

If the compounds are basic, salts could be prepared in a form ofpharmaceutically acceptable salts that will be prepared from nontoxicinorganic or organic acids including but not limited to hydrochloric,hydrobromic, phosphoric, sulfuric, tartaric, citric, acetic, fumaric,alkylsulphonic, naphthalenesulphonic, para-toluenesulphonic, camphoricacids, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, gluconic, glutamic, isethonic, lactic, maleic, malic,mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic,phosphoric, and succinic.

While it may be possible for the compounds to be administered as the rawchemical, it is also possible to present them as a pharmaceuticalformulation. Accordingly, the present invention provides apharmaceutical formulation comprising a compound or a pharmaceuticallyacceptable salt, ester, prodrug or solvate thereof, together with one ormore pharmaceutically acceptable carriers thereof and optionally one ormore other therapeutic ingredients. The carrier(s) must be “acceptable”in the sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof. Properformulation is dependent upon the route of administration chosen. Any ofthe well-known techniques, carriers, and excipients may be used assuitable and as understood in the art; e.g., in Remington'sPharmaceutical Sciences. The pharmaceutical compositions of the presentinvention may be manufactured in a manner that is itself known, e.g., bymeans of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or compressionprocesses.

The formulations include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous, intraarticular,and intramedullary), intraperitoneal, transmucosal, transdermal, rectaland topical (including dermal, buccal, sublingual and intraocular)administration although the most suitable route may depend upon forexample the condition and disorder of the recipient. When used in thediagnostic imaging methods of the invention, compounds can beadministered to the patient (e.g., a human) by intravenous injection.The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing into association a compound ofthe present invention or a pharmaceutically acceptable salt, ester,prodrug or solvate thereof (“active ingredient”) with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both and then, if necessary, shaping the product intothe desired formulation.

Formulations suitable for oral administration may be presented asdiscrete units such as capsules, cachets or tablets each containing apredetermined amount of the active ingredient; as a powder or granules;as a solution or a suspension in an aqueous liquid or a non-aqueousliquid; or as an oil-in-water liquid emulsion or a water-in-oil liquidemulsion. The active ingredient may also be presented as a bolus,electuary or paste.

Pharmaceutical preparations which can be used orally include tablets,push-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. Tablets maybe made by compression or molding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such as apowder or granules, optionally mixed with binders, inert diluents, orlubricating, surface active or dispersing agents. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredient therein. All formulationsfor oral administration should be in dosages suitable for suchadministration. The push-fit capsules can contain the active ingredientsin admixture with filler such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds may be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers may be added.Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Compounds may be formulated for parenteral administration by injection,e.g., by bolus injection or continuous infusion. Formulations forinjection may be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. Compositions may takesuch forms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. The formulations may be presentedin unit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in powder form or in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example, saline or sterile pyrogen-free water,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Formulations for parenteral administration include aqueous andnon-aqueous (oily) sterile injection solutions of the active compoundswhich may contain antioxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

In addition to the formulations described previously, a compound mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, acompound may be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

For buccal or sublingual administration, a compound may take the form oftablets, lozenges, pastilles, or gels formulated in conventional manner.Such compositions may comprise the active ingredient in a flavored basissuch as sucrose and acacia or tragacanth.

A compound may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter, polyethylene glycol, or otherglycerides.

A compound may be administered topically, that is by non-systemicadministration. This includes the application of a compound externallyto the epidermis or the buccal cavity and the instillation of such acompound into the ear, eye and nose, such that the compound does notsignificantly enter the blood stream. In contrast, systemicadministration refers to oral, intravenous, intraperitoneal andintramuscular administration.

Formulations suitable for topical administration include solid, liquidor semi-liquid preparations suitable for penetration through the skin tothe site of inflammation such as gels, liniments, lotions, creams,ointments or pastes, and drops suitable for administration to the eye,ear or nose. The active ingredient may comprise, for topicaladministration, from 0.001% to 10% w/w, for instance from 1% to 2% byweight of the formulation. It may however comprise as much as 10% w/wbut preferably will comprise less than 5% w/w, more preferably from 0.1%to 1% w/w of the formulation.

Via the topical route, a pharmaceutical composition may be in the formof liquid or semi liquid such as ointments, or in the form of solid suchas powders. It may also be in the form of suspensions such as polymericmicrospheres, or polymer patches and hydrogels allowing a controlledrelease. This topical composition may be in anhydrous form, in aqueousform or in the form of an emulsion. The compounds are used topically ata concentration generally of between 0.001% and 10% by weight andpreferably between 0.01% and 1% by weight, relative to the total weightof the composition.

For administration by inhalation, a compound can be convenientlydelivered from an insufflator, nebulizer pressurized packs or otherconvenient means of delivering an aerosol spray. Pressurized packs maycomprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Alternatively, for administration by inhalation or insufflation, acompound may take the form of a dry powder composition, for example apowder mix of the compound and a suitable powder base such as lactose orstarch. The powder composition may be presented in unit dosage form, infor example, capsules, cartridges, gelatin or blister packs from whichthe powder may be administered with the aid of an inhalator orinsufflator.

Preferred unit dosage formulations are those containing an effectivedose, as herein below recited, or an appropriate fraction thereof, ofthe active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above, formulations described herein may include other agentsconventional in the art having regard to the type of formulation inquestion, for example those suitable for oral administration may includeflavoring agents.

A compound may be administered orally or via injection at a dose of from0.1 to 500 mg/kg per day. The dose range for adult humans is generallyfrom 5 mg to 2 g/day. Tablets or other forms of presentation provided indiscrete units may conveniently contain an amount of compound which iseffective at such dosage or as a multiple of the same, for instance,units containing 5 mg to 500 mg, usually around 10 mg to 200 mg.

Compounds can be administered at a daily dose of about 0.001 mg/kg to100 mg/kg of body weight, in 1 to 3 dosage intakes. Further, compoundscan be used systemically, at a concentration generally of between 0.001%and 10% by weight and preferably between 0.01% and 1% by weight,relative to the weight of the composition.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration.

A compound can be administered in various modes, e.g. orally, topically,or by injection. The precise amount of compound administered to apatient will be the responsibility of the attendant physician. Thespecific dose level for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex, diets, time ofadministration, route of administration, rate of excretion, drugcombination, the precise disorder being treated, and the severity of theindication or condition being treated. Also, the route of administrationmay vary depending on the condition and its severity.

In certain instances, it may be appropriate to administer at least onecompound described herein (or a pharmaceutically acceptable salt, ester,or prodrug thereof) in combination with another therapeutic ordiagnostic agent. By way of example only, if one of the side effectsexperienced by a patient upon receiving one of the compounds describedherein is hypertension, then it may be appropriate to administer ananti-hypertensive agent in combination with the initial therapeuticagent. Or, by way of example only, the therapeutic effectiveness of oneof the compounds described herein may be enhanced by administration ofan adjuvant (i.e., by itself the adjuvant may only have minimaltherapeutic benefit, but in combination with another therapeutic agent,the overall therapeutic benefit to the patient is enhanced). Or, by wayof example only, the benefit of experienced by a patient may beincreased by administering one of the compounds described herein withanother therapeutic agent (which also includes a therapeutic regimen)that also has therapeutic benefit. By way of example only, in atreatment for pain involving administration of one of the compoundsdescribed herein, increased therapeutic benefit may result by alsoproviding the patient with another therapeutic agent for pain. In anycase, regardless of the disease, disorder or condition being treated,the overall benefit experienced by the patient may simply be additive ofthe two therapeutic agents or the patient may experience a synergisticbenefit.

Specific, non-limiting examples of possible combination therapiesinclude use of a compound together with inert or active compounds, orother drugs including wetting agents, flavor enhancers, preservingagents, stabilizers, humidity regulators, pH regulators, osmoticpressure modifiers, emulsifiers, UV-A and UV-B screening agents,antioxidants, depigmenting agents such as hydroquinone or kojic acid,emollients, moisturizers, for instance glycerol, PEG 400, or urea,antiseborrhoeic or antiacne agents, such as S-carboxymethylcysteine,S-benzylcysteamine, salts thereof or derivatives thereof, or benzoylperoxide, antibiotics, for instance erythromycin and tetracyclines,chemotherapeutic agent, for example, paclitaxel, antifungal agents suchas ketoconazole, agents for promoting regrowth of the hair, for example,minoxidil (2,4-diamino-6-piperidinopyrimidine 3-oxide), non-steroidalanti-inflammatory agents, carotenoids, and especially p-carotene,antipsoriatic agents such as anthralin and its derivatives,eicosa-5,8,11,14-tetraynoic acid and eicosa-5,8,11-triynoic acid, andesters and amides thereof, retinoids, e.g., RAR or RXR receptor ligands,which may be natural or synthetic, corticosteroids or oestrogens,alpha-hydroxy acids and a-keto acids or derivatives thereof, such aslactic acid, malic acid, citric acid, and also the salts, amides oresters thereof, or p-hydroxy acids or derivatives thereof, such assalicylic acid and the salts, amides or esters thereof, ion-channelblockers such as potassium-channel blockers, or alternatively, moreparticularly for the pharmaceutical compositions, in combination withmedicaments known to interfere with the immune system, anticonvulsantagents include, and are not limited to, topiramate, analogs oftopiramate, carbamazepine, valproic acid, lamotrigine, gabapentin,phenytoin and the like and mixtures or pharmaceutically acceptable saltsthereof. A person skilled in the art will take care to select the othercompound(s) to be added to these compositions such that the advantageousproperties intrinsically associated with the compounds are not, or arenot substantially, adversely affected by the envisaged addition.

In any case, the multiple therapeutic or diagnostic agents may beadministered in any order or even simultaneously. If simultaneously, themultiple therapeutic or diagnostic agents may be provided in a single,unified form, or in multiple forms (by way of example only, either as asingle pill or as two separate pills). One of the therapeutic ordiagnostic agents may be given in multiple doses, or both may be givenas multiple doses. If not simultaneous, the timing between the multipledoses may be any duration of time ranging from a few minutes to fourweeks.

Thus, in another aspect, methods for diagnosing or treating diseases,disorders, conditions, or symptoms in a subject (e.g., a human oranimal) in need of such treatment are presented herein, the methodscomprising the step of administering to the subject an amount of acompound effective to reduce or prevent the disease, disorder,condition, or symptom, in combination with at least one additional agentfor the treatment of said disorder that is known in the art.

EXAMPLES

It is understood that the foregoing examples are merely illustrative ofthe present invention. Certain modifications of the articles and/ormethods employed may be made and still achieve the objectives of theinvention. Such modifications are contemplated as within the scope ofthe claimed invention.

Example 1 Gene Array Analysis Before and after TBI in Rodent Model

System x_(c) ⁻ is a cystine/glutamate antiporter comprised of twodistinct subunits xCT and 4F2hc (SLC3A2) and a member of the heteromericamino acid transporter (HAT) family. Under physiological conditions,System x_(c) ⁻ mediates the exchange of extracellular L-cystine andintracellular L-glutamate across the plasma membrane. In the CNS, theinflux of L-cystine represents the critical rate limiting step in thebiosynthesis of glutathione (GSH) while the concurrent efflux ofL-glutamate serve as a non-vesicular route of excitatoryneurotransmitter release to initiate excitatory amino acid (EAA)signalling. GSH serves as the key cellular antioxidant responsible forscavenging reactive oxygen species (ROS) that develop as a result ofphysiological cellular metabolism. Thus a global loss of System x_(c) ⁻activity would result in decreased intracellular glutathione levels,leaving the CNS vunerable to oxidative stress due to an increase incellular ROS. While it is likely that other antioxidant systems such asSOD1, SOD2, and catalase would initially metabolize ROS, as anindividual ages these compenstatory enzymes lose scavenging efficiencyresulting in a prolonged elevation in ROS. With glutathione missing andsupporting antioxidant systems operating with less efficiency, ROSaccumulation could result in unsurmountable oxidative stress leading toneuropathology associated with the gradual process of neurodegenerativeevents leading to CTE.

In animal studies we have found that a single TBI produced a rapid,global, long-term loss of a key transporter protein subunit, xCT(SLC7A11; FIGS. 1-3). Over time (46 days post-TBI), the levels of xCTgradually returned but never reached pre-TBI levels suggesting theinjury induced a long-term loss of xCT. xCT is the catalytic subunit ofSystem xc−, a ubiquitous antiporter responsible for the biosynthesis ofgluthathione (GSH) in the brain. GSH is the primary cellularanti-oxidant that scavenges damaging reactive oxygen species (ROS) thatdevelop as a result of normal metabolism or neuronal injury.

Example 2 miRNA Expression Profiles of Oxidative Stress and AntioxidantDefense Genes

To follow up this study, we performed a gene array analysis to determineif oxidative stress genes up-regulate to compensate for the loss of GSH(FIG. 4). From this study we found exactly the opposite occurred; TBIresulted in a down-regulation of key anti-oxidant defense genes leavingneurons critically susceptible to ROS damage. In an effort to elucidatethe mechanism of how the xCT subunit and anti-oxidant defense genes weredown-regulated by TBI we performed a microRNA (miRNA) expression profilestudy on rat (R. norvegus) cortical brain tissue following neuronalinjury in a lateral fluid percussion TBI model (FIG. 5). In light of ourprior findings, we chose miRNAs that specifically interact withanti-oxidant defense genes and xCT. A specific cluster of miRNAs thatwere significantly (2-13 fold) up or down regulated as a result of TBIwas discovered (Table 3). Further studies indicate all of these highlyconserved miRNAs are novel to TBI research, and can be detected in theplasma.

TABLE 3 miRNA Name Fold Up-Regulation rno-miR-142-3p 2.0894rno-miR-21-5p 1.7652 rno-let-7a-5p 1.7782 rno-let-7b-5p 1.6374rno-let-7f-5p 3.9548 rno-miR-144-3p 20.49 rno-miR-150-5p 8.8712rno-miR-32-5p 1.9359 rno-miR-130a-3p 1.5581 rno-miR-101a-3p 1.8134rno-miR-18a-5p 1.8539 rno-let-7d-5p 1.8442 rno-miR-181b-5p 1.5577rno-miR-223-3p 3.3426 rno-miR-320-3p 1.6849 rno-miR-374-5p 1.8993rno-let-7e-5p 1.9764 rno-miR-196b-5p 8.9494 rno-miR-96-5p 1.6113rno-miR-423-3p 2.8863 rno-miR-210-3p 1.7867 rno-miR-182 2.7308rno-miR-196a-5p 12.2571 cel-miR-39-3p 6.3604 cel-miR-39-3p 6.9183 FoldDown-Regulation rno-miR-9a-5p −220228.6761

Example 3 miRNA Expression Profiles of Human Peripheral Blood Plasma

Approximately 3 mL of blood was collected from each subject andprocessed as described below. Total RNA was isolated and prepared from200 μL plasma according to the miRNeasy Serum/Plasma Kit (50) protocolaccording to the manufacturer's instructions (Qiagen). cDNA was preparedusing the isolated total RNA. Real-time PCR was performed using theQiagen miScript SYBR Green PCR kit and custom miScript miRNA PCR Arrayon a Bio-Rasd iQ5 cycler. Data analysis was performed using the QiagenData Analysis Center. The following plasma samples were obtained:

Sample Source Description n = Control No TBI, non-athlete 14 FootballPlayers No TBI with 3 months 49 Soccer Players No TBI with 3 months 19Acute TBI 24-72 post-TBI 4 Chronic TBI 3 months post-TBI 6All plasma samples were obtained by voluntary donation according to theInstitutional Review Board protocols of The University of Montana.Samples were obtained at random (control group), from student athletesparticipating in football or soccer sports, and from subjects known tohave suffered acute (within 72 hours) TBI or chronic (greater than 72hours) TBI.

The data presented shows the fold change when comparing sample, the 95%CI and p values. Initial analysis of the data show that there are strongtrends towards increases in some miRNA levels following Acute TBI withmiR-142-3p, miR-150-5p, and miR-196b-5p showing significance in thescreening between Control and Acute TBI. Significant differences werealso found in let-7f-5p, miR-150-5p, and miR-196b-5p between Control andSoccer Players. Much of the data is trending toward significance (i.e.,p<0.05). The data also suggest there may be a strong influence of genderon the changes in miRNA levels (Football v Soccer Players).Interestingly the analysis of the chronic TBI group suggests that thelevels of miRNA rebound and drop potentially as a compensatory measureand this finding suggests we may able to further use this panel todistinguish between acute and chronic TBI using this panel.

All Embodiments

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

Other embodiments are within the claims.

What is claimed is:
 1. A method of detecting a brain injury in a patientcomprising the steps of contacting a biological sample derived from apatient with at least one miR-specific oligodeoxynucleotide probe havingat least 70% complementarity to a sequence selected from SEQ ID NOs.1-69; determining the expression level of at least one microRNArepresented by SEQ ID NOs. 1-69 by quantifying said at least onemiR-specific oligodeoxynucleotide probe; and comparing said expressionlevel with a control expression level derived from a healthy subject;wherein a 1.2 fold or greater difference between said patient andcontrol microRNA expression levels indicates that said patient hassuffered a brain injury.
 2. The method of claim 1 further comprising thestep of treating said patient with a therapeutically-effective amount ofan antioxidant if a brain injury is detected.
 3. The method of claim 2,wherein said antioxidant is selected from the group consisting ofalpha-tocopherol, ascorbate, coenzyme Q, alpha-lipoic acid, curcumin,glutathione, uric acid, a carotene, superoxide dismutase, a catalase, aperoxiredoxin, a thioredoxin, tirilazad mesylate, and NXY-059.
 4. Themethod of claim 1, wherein said patient is a human.
 5. The method ofclaim 1, wherein said biological sample is blood, cerebral spinal fluid,or brain tissue.
 6. The method of claim 1, wherein said biologicalsample is blood plasma or serum.
 7. The method of claim 1, wherein saidbrain injury is a traumatic brain injury (TBI) or chronic traumaticencephalopathy.
 8. The method of claim 1, wherein said measuringcomprises polymerase chain reaction (PCR), in situ hybridization,Northern blot, or gene chip analysis.
 9. The method of claim 1, whereinsaid miR-specific oligodeoxynucleotide probe comprises DNA.
 10. Themethod of claim 1, wherein said microRNA control expression level isderived from a sample derived from said patient prior to sustaining abrain injury.
 11. The method of claim 1, wherein said biological sampleis derived from said patient within seventy two (72) hours of sustaininga suspected brain injury.
 12. The method of claim 1 repeated onbiological samples derived from said patient over a period of time,wherein changes in said microRNA expression levels over time indicateprogression or regression of said brain injury.
 13. The method of claim1, wherein said at least one miR-specific oligodeoxynucleotide probe hasat least 90% complementarity to a sequence selected from SEQ ID NOs.1-69.
 14. A minimally-invasive method of detecting a brain injury in apatient comprising the steps of contacting a blood, plasma, or serumsample derived from a patient with at least one miR-specificoligodeoxynucleotide probe having at least 70% complementarity to asequence selected from SEQ ID NOs. 1-69; determining the expressionlevel of at least one microRNA represented by SEQ ID NOs. 1-69 byquantifying said at least one miR-specific oligodeoxynucleotide probe;and comparing said expression level with a control expression levelderived from a healthy subject; wherein a 1.2 fold or greater differencebetween said patient and control microRNA expression levels indicatesthat said patient has suffered a brain injury.
 15. The method of claim14 further comprising the step of treating said patient with atherapeutically-effective amount of an antioxidant if a brain injury isdetected.
 16. A kit for detecting a brain injury, the kit comprising (a)one or more miR-specific oligonucleotide probes having at least 70%complementarity to a sequence selected from SEQ ID NOs. 1-69, (b) one ormore control samples, and (c) instructions indicating the use of saidprobes and said control samples for detecting a brain injury.